The fragile X syndrome is the most common form of inherited mental retardation in humans and is estimated to afflict roughly 1 in 2500 males and 1 in 5000 females. Conditions associated with the syndrome include mild to moderate cognitive abnormalities, as well as behavioral disorders similar to autism, attention deficit disorder, obsessive-compulsive tendencies, hyperactivity, slow development of motor skills, and anxiety fear disorder. The syndrome is also frequently accompanied by seizures, by macroorchidism, and by subtle craniofacial dysmorphia.
Studies have shown that fragile X syndrome results from a deficiency of the fragile X mental retardation protein, FMRP, which is encoded by the X-linked FMR1 gene. Fragile X syndrome usually results from the transcriptional silencing of this gene brought about by the expansion and hypermethylation of a (CGG)n trinucleotide repeat in the gene's 5′ untranslated region (UTR), indicating that the presence of FMRP is essential for higher cognitive function.
Cloning of the FMR1 gene led to the finding that FMRP is an RNA binding protein, with sequence analysis indicating a number of putative RNA binding domains in FMRP, specifically two tandem KH domain sequences and a C-term-inal RGG box sequence. In addition, FMRP has both a nuclear localization signal and a Rev-like export signal and is consequently believed to shuttle in and out of the nucleus. Despite this ability, the protein is largely cytoplasmic, and is incorporated into large messenger-ribonucleoprotein (mRNP) particles containing other RNA-binding proteins such as the autosomal paralogs of FMRP, FXR1P and FXR2P, which are encoded by fragile X-related genes FXR1 and FXR2, respectively. In the cytoplasm, FMRP-mRNP is normally associated with translating polyribosomes, although mutations that alter this association have been identified. A missense mutation in the second KH domain of FMRP (I304N), for example, prevents this polyribosome association and also results in a severe fragile X phenotype.
Although the exact mode of action of FMRP is not precisely understood, one unifying model for FMRP function is that the protein shuttles specific mRNAs from the nucleus to postsynaptic sites, where the mRNAs are held in a translationally inactive form until synaptic input alters FMRP activity to allow their translation. Thus, when FMRP is absent, the mRNAs normally associated with FMRP-mRNP complexes may be translationally misregulated, which, in the brain, leads to cognitive deficits.
Although FMRP has been demonstrated to be an RNA binding protein, to date there has been little success at identifying the particular RNAs or mRNAs to which FMRP binds. This inability to identify the RNAs upon which FMRP acts has left a critical gap in understanding how the absence of FMRP leads to mental retardation. There is thus a long felt need for identifying and characterizing the RNA targets of FMRP.